【作者】 白燕；

【导师】 金润铭；

【作者基本信息】 华中科技大学 ， 儿科学， 2008， 博士

【摘要】 第一部分人巨细胞病毒感染与特发性血小板减少性紫癜患儿细胞免疫功能及治疗转归的临床研究目的探讨人类巨细胞病毒（HCMV）感染与儿童特发性血小板减少性紫癜（ITP）发病、疗效及预后的相关性,为临床更合理有效治疗ITP提供部分参考。方法用ELISA法和流式细胞仪分别检测患儿血清HCMV抗体及外周血淋巴细胞亚群,比较2002年1月-2006年12月在本院儿科就诊的230例ITP患儿及对照组200例同期急性上呼吸道感染患儿的HCMV感染率及细胞免疫状态。进一步将ITP患儿按有无HCMV感染分为HCMV感染组和非感染组,比较二组患儿细胞免疫状态、近期（二周）治疗效果、远期（大于6个月）疾病转归之间的关系。结果230例ITP患儿中,伴HCMV感染者88例（38.26%）,淋巴细胞亚群比例异常165例（71.74%）;同期200例急性上呼吸道感染患儿中HCMV感染22例（11.0%）,淋巴细胞亚群比例异常36例（18.0%）,二组比较有显著统计学差异（P＜0.01,P＜0.01）。初诊时HCMV感染组细胞免疫异常74例（84.09%）,非感染组91例（64.08%）;6个月后,HCMV感染组检测到细胞免疫异常24例（27.27%）,非感染组5例（3.52%）。二组比较有显著统计学差异（P＜0.01）。HCMV感染组与非感染组近期治疗效果无统计学差异（P＞0.05）,HCMV感染组中35例（39.78%）病程超过6个月,而非感染组中24例（16.90%）病程超过6个月,二组比较有显著统计学差异（P＜0.01）。抗HCMV治疗组54例,病程超过6个月15例（27.78%）,常规治疗组34例,病程超过6个月20例（58.82%）,二组相比有显著统计学差异（P＜0.01）。结论HCMV感染诱发的细胞免疫异常是儿童ITP发病及病程迁延的重要因素。抗HCMV治疗对于ITP的恢复有明确效果,且副作用少,患儿耐受性好。第二部分儿童AITP治疗前后CD4⁺CD25^HighFoxp3⁺Treg细胞与Foxp3基因表达的变化目的通过检测静注丙种球蛋白、肾上腺皮质激素（简称激素）治疗前后儿童急性特发性血小板减少性紫癜（AITP）CD4⁺CD25^HighFoxp3⁺Treg细胞比例及Foxp3基因表达的变化,探讨CD4⁺CD25^HighFoxp3⁺Treg细胞在AITP发病中的作用及对静注丙种球蛋白、激素治疗的效应关系。方法流式细胞仪检测AITP患儿治疗前后CD4⁺CD25^HighFoxp3⁺Treg细胞比例变化,RT-PCR法检测外周血单个核细胞中Foxp3mRNA的表达。结果AITP患儿治疗前CD4⁺CD25^HighFoxp3⁺Treg细胞比例与Foxp3基因表达水平明显低于正常对照组（P＜0.01）;单用激素或联用激素和静注丙种球蛋白治疗后AITP患儿CD4⁺CD25（High）Foxp3⁺Treg细胞比例和Foxp3基因表达水平较治疗前明显增高（P＜0.01）,其中联用静注丙种球蛋白和激素组的增高水平更为明显（P＜0.01）;联用静注丙种球蛋白和激素治疗的AITP患儿血小板恢复正常所需的时间比单用激素组明显缩短（P＜0.05）。结论CD4⁺CD25^HighFoxp3⁺Treg细胞比例下降及Foxp3基因表达降低是AITP的发病机制之一,丙种球蛋白和激素可以通过诱导CD4⁺CD25^HighFoxp3⁺Treg的扩增及活化发挥治疗AITP的作用。第三部分儿童特发性血小板减少性紫癜淋巴毒素基因多态性分析目的研究中国湖北地区特发性血小板减少性紫癜患儿LTα+252位点基因多态性的分布,探讨LTα不同基因型与ITP发病易感性的可能关系。方法用PCR—RFLP法检测88例ITP患儿与100例健康儿童LTα+252位点基因的多态性,比较两组患儿不同基因型出现的频率。结果ITP患儿LTα+252位点G/G、G/A、A/A基因型频率分别为27.27%、50.00%、22.72%,与正常儿童相比无明显统计学差异（P＞0.05）。结论中国湖北地区儿童LTα+252位点基因多态性与ITP发病无明显相关性。更多还原

【Abstract】 PartⅠClinic Study on Cell Immune Conditions and Curative Turnover of Idiopathic Thrombocytopenic Purpura with Human Cyto-megalovirus Infection in ChildrenObjective Exploring the correlation between HCMV infection and the onset, therapeutic effect and prognosis in children with idiopathic thrombocytopenic purpura（ITP）, in order to give some reference for more effective treatment of this disease.Methods Comparing the HCMV infection rate and cell immune conditions between 230 children with ITP and the control group of 200 children with acute upper respiratory infection（AURI） treated in our department between January,2002 to December,2006,by testing the serum HCMV antibodies with ELISA method and detecting the peripheral blood lymohocyte subsets with flow cytometry individually.Further,ITP patients were divided into two groups based on whether they were infected with HCMV or not..The two groups were compared with regard to the cell immune conditions,short-term effectiveness of treatment（two weeks） and long-term results（over six months）.Results 88 out of 230（38.26%） ITP children were found with HCMV infection,and 165 cases（71.74%） with abnormal peripheral blood lymphocyte subgroup.22 out of 200 （11.0%）AURI patients were found with HCMV infection,and 36 cases（18.0%） with abnormal lymphocyte subgroup.There was a significant difference between these two groups（P＜0.01）.At the first visit,74（84.09%） were found with abnormal cell immune condition in the HCMV group,and 91（64.08%） in non-infected group;after 6 months, 24（27.27%） were found abnormal in the HCMV group,and 5（3.52%） in non-infected ones. There was a remarkable difference between these two groups（P＜0.01）.No significant difference existed in the short-term effectiveness of treatment between the group with HCMV infection and the group without that（P＞0.05）.35 cases（39.78%） in the HCMV group had a course over 6 months,whereas 24（16.90%） in non-infected group.The difference is significant（P＜0.01）.20 out of 34（58.82%） had a course over 6 months in the regular therapy group,and 15 out of 54（27.78%） did in the antivirus therapy group.The difference is also significant（P＜0.01）.Conclusion Abnormal cell immune conditions induced by HCMV infection is probably one of the important factors that make a prolonged course of ITP in children. Further more,our study showed that anti-HCMV therapy resulted in good recovery of children from ITP with little side effect and that most children tolerated well to the therapy. PartⅡThe change of CD4+CD25HighFoxp3+Treg cell and the Foxp3 gene expression in childhood acute idiopathic thrombocytopenic purpura before and after treatmentObjective Exploring the CD4⁺CD25^HighFoxp3⁺Treg cell function in the mechanism of acute idiopathic thrombocytopenic purpura and the role in therapy with gamma globulin and adrenal corticoid by studying the change of CD4⁺CD25^HighFoxp3⁺Treg cell percentage and the Foxp3 gene expression before and after treatment of acute idiopathic thrombocytopenic purpura.Method The change of CD4⁺CD25^HighFoxp3⁺Treg cell percentage was detected by flow cytometry,while the expression of Foxp3 gene was tested by RT-PCR.Results The CD4⁺CD25^HighFoxp3⁺Treg cell percentage and the Foxp3 gene expression in acute idiopathic thrombocytopenic purpura group before treatment were significantly lower than those of the normal control group（P＜0.01）,but after treatment with adrenal corticoid alone or plus gamma globulin,the CD4⁺CD25^HighFoxp3⁺Treg cell percentage and the Foxp3 gene expression in acute idiopathic thrombocytopenic purpura group rose obviously higher than those of the normal control group（P＜0.01）.Compared with group treated with adrenal corticoid alone,the group treated with corticoid plus gamma globulin was remarkly higher in CD4⁺CD25^HighFoxp3⁺Treg cell percentage and the expression of Foxp3 gene.Meanwhile,the recovery time of platelet number in group treated with corticoid plus gamma globulin was significantly shorter than that of using corticoid only（P＜0.05）.Conclusion The decreasing of CD4⁺CD25^HighFoxp3⁺Treg cell and the Foxp3 gene expression play a role in the mechanism of children acute idiopathic thrombocytopenic purpura.Furthermore,adrenal corticoid and gamma globulin have a therapeutic effect on acute idiopathic thrombocytopenic purpura by inducing the expansion and activation of CD4⁺CD25^HighFoxp3⁺Treg cell. PartⅢAnalysis of lymphotoxin gene polymorphism in children with idiopathic thrombocytopenic purpuraObjective To study on the gene polymorphism of LTα+252 distribution in children with ITP in Hubei,China,and explore the association of genotype frequency of LTαgene with the susceptivity of ITP.Method Eighty- eight ITP children and one hundred health controls were genotyped by the PCR-RFLP method for gene polymorphism of LTα+252,then compared the genotypes frequency between these two groups.Result The genotypes frequency of LTα+252G/G、G/A、A/A in ITP children were 27.27%、50.00%、22.72%individually,and there were no statistic difference compared with the control group.Conclusion No association was detected between the heterozygous genotype of LTα+252 and ITP in children of Hubei,China.更多还原